Scientists at Transkaryotic Therapies Inc. (TKT) have developeda system to isolate disease-associated genes that is apparently10 to 100 times faster than current technologies. Dubbed"recombination walking," the new technology is reported byAllan Miller and his colleagues in the latest issue of theProceedings of the National Academy of Sciences under the title"Recombination Walking: Genetic Selection of Clones FromPooled Libraries of Yeast Artificial Chromosomes byHomologous Recombination."
The trick is to pinpoint the location of disease-causing genes ontheir chromosomes so they can be isolated and analyzed. Thistask is not so difficult when one knows what protein thedefective gene makes. But many disease-causing genes aredefined by the disease per se: the protein or proteins are notknown. There is a general approach that scientists haveadopted for honing in on a gene's chromosomal location: Itstarts with an analysis of the pedigree of families exhibiting acertain disease, then proceeds to a rough mapping procedure(using restriction fragment length polymorphisms or RFLPs asmarkers) that places the putative disease gene in a particularsegment of a particular chromosome.
But "you still have to figure out in relation to that markerexactly where the gene is," explained Richard Selden, TKT'sfounder and chief scientific officer. "The marker could be 10million base pairs away from the gene." To get closer, scientiststhen construct a series of clones containing smaller segments ofthat fragment -- say 50,000 base pairs each --with overlappingends that allow them to gradually "walk along thechromosome" until they reach the segment of DNA that's "righton top of the gene," Selden added. That one segment, then,which may contain 50,000 base pairs, can be cloned and thegene fished out.
But the whole procedure takes time and money -- and lots ofboth. According to Selden, it took dozens of individuals workingfor six to eight years to walk two million base pairs to land onthe gene for cystic fibrosis. And the price tag topped $100million. It's quicker today -- the same procedure would nowtake one full-time worker six weeks to complete -- but that'sstill too slow.
One way researchers have devised to speed up the process is touse yeast artificial chromosomes (YACs) as cloning vehicles.YACs allow DNA fragments that are 100 to 2,000 kilobases longto be propagated -- much longer than the fragments used inearlier types of cloning procedures. This maximizes thedistance covered by each walking step. Still, one ends up with50,000 to 100,000 candidate YACs; all of them need to bescreened manually by hybridization methods to pick out theone YAC that contains the gene of interest.
But recombination walking, explained TKT's Selden, speeds upeven that procedure. It's based on the specificity of geneticrecombination in yeast, and permits the yeast to actuallyperform the screening. Researchers introduce a target DNAsequence into the YACs, each of which contains a differentpiece of genomic DNA. Any individual YACs that contain asequence that matches with the target DNA take it up oracquire it by homologous recombination. When a selectablemarker (for antibiotic resistance, for instance) is included, it'spossible to fish out that one YAC in 100,000 that contains thegenetic sequences of interest. The whole procedure "takesabout two hours' effort by one person, and 20 runs can be donesimultaneously," Selden said.
Selden views TKT's recombination walking technology as beingcomplementary to the genome mapping and sequencing workbeing performed as part of the human genome project.
The company's initial disease targets are diabetes, Alzheimer'sdisease and malignant melanoma.
-- Jennifer Van Brunt Senior Editor
(c) 1997 American Health Consultants. All rights reserved.